Lyme disease, the most common arthropod-borne disease in the United States, is caused by infection with the spirochete Borrelia burgdorferi. The lack of a system to genetically manipulate B. burgdorferi has hindered attempts to study its biology and pathogenesis on a molecular genetic level. Several components are required to create a useful genetic system for assaying the effect of individual genes. These include selectable markers, stable plasmid vectors, and a method of transformation. This project proposes to develop a genetic system for B. burgdorferi, initially based on electroporation and resistance to the antibiotic coumermycin A1. This marker will be used to inactivate genes by insertion and to select for the maintenance of broad host range vectors and recombinant natural plasmids. In addition, other selectable genetic markers will be screened for use in the borrelia system. Establishment of such a system will facilitate identifying gene defects in mutant strains by complementation and will expedite the study of plasmid replication. Construction of several vectors with different antibiotic resistance markers will permit the inactivation of genes in vitro, replacement of the wild type locus in vivo, and complementation of the defect. These genetic techniques will be the first steps in systematically dissecting the genes that are required for growth and pathogenesis.